Cellular context in epigenetics: Quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets

Epigenetics in general and microRNA (miRNA) in particular are an important and growing field of research, and while significant advances in the role of miRNA in a variety of diseases including cancer have been made, the majority of the information on the relationship between miRNA and its putative target proteins have been made on homogenized tissue, which, while useful, provides no information on tissue- or cell-specific signatures or relationships. Developments in spectral imaging have made it possible to image and quantitate samples labeled for both a miR and its putative target, using either brightfield or fluorescence labeling. Advances in automated morphometric and cellular quantitation enable the extraction of quantitative, tissue-specific measures of marker in each cell of a tissue section. This per-cell quantitative data can then be displayed as scatter plots, in a manner analogous to flow cytometry data, and multimarker phenotypes can be determined from threshold-based quadrant analysis. This paper describes the utility of the Nuance™ multispectral imaging system and the inForm™ image analysis software for assessing morphological distributions of miRNAs and their putative targets in thin tissue sections, and provides methodologies for the per-cell quantitative analysis of both markers.